This invention relates to antibodies and antigen binding fragments thereof which bind to PSGL-1, methods of their use in treating inflammatory and thrombotic conditions, and methods of screening for PSGL-1 inhibitory substances.
Selectins and P-Selectin Glycoprotein Ligand-1 (PSGL-1).
The body regulates inflammatory responses through a series of multistep adhesive and signaling events in response to infection and injury [1-3]. To start this process circulating leukocytes must first adhere to the vascular wall under the shear forces of flow. The selectins mediate this first adhesive step which is characterized by tethering and rolling of leukocytes on endothelial cells, platelets, or other leukocytes [4, 5]. P-selectin is expressed on activated platelets and endothelial cells and binds to ligands on most leukocytes. L-selectin is expressed on most leukocytes and binds to ligands on some endothelial cells and on other leukocytes. E-selectin is expressed on cytokine-activated endothelial cells and also binds to ligands on most leukocytes. The expression of the selectins and their ligands is tightly regulated to initiate and terminate the inflammatory response. Inappropriate expression of these molecules contributes to leukocyte-mediated tissue damage in many inflammatory and thrombotic disorders [6].
All three selectins are type 1 membrane glycoproteins with an NH2-terminal C-type lectin domain, an EGF-like domain, a varying series of short consensus repeats, a transmembrane domain, and a short cytoplasmic tail. Selectins mediate cell-cell adhesion through interactions of the lectin domains with specific glycoconjugate ligands. The selectins bind with low affinity to the tetrasaccharide sialyl Lewis x (sLex, NeuAcα2,3Galβ1,4[Fucα1,3]GlcNAc) and its isomer sialyl Lewis a (sLea; NeuAcα2,3Galβ1,3[Fucα1,4]GlcNAc). P- and L-selectins, but not E-selectin, also bind to particular sulfated carbohydrates that lack sialic acid and fucose, such as heparan sulfate [4, 5, 7]. Selectins bind with higher affinity or avidity to only a few glycoproteins. Most are mucins, which are glycoproteins with multiple Ser/Thr-linked oligosaccharides (O-glycans) and repeating peptide motifs [4, 5]. Crystal structures of sLex bound to the lectin domains of P- and E-selectin showed a network of interactions between fucose, a single Ca2+ ion and several amino acids including those that coordinate the Ca2+ [8]. The sialic acid and galactose also interact with the lectin domain. Targeted disruption of the gene encoding α1,3 fucosyltransferase (Fuc-TVII) in mice significantly reduces selectin-mediated leukocyte trafficking. Disruption of the genes for both Fuc-TVII and Fuc-TIV completely eliminates these interactions [9, 10], indicating that all physiologically relevant selectin ligands require α1,3-linked fucose.
PSGL-1 is the Specific Glycoprotein Ligand for P-Selectin on Leukocytes.
PSGL-1 (CD162) has been shown to be the specific ligand for P-selectin. Early studies using ligand blotting and affinity chromatography showed that P-selectin binds preferentially to a single glycoprotein in human myeloid cells [11]. The glycoprotein, (now known as P-selectin glycoprotein ligand-1, or PSGL-1), was shown to be a disulfide-bonded homodimer with two 120-kD subunits by SDS-PAGE under reducing and nonreducing conditions. Digestion with peptide N-glycosidase F demonstrated that PSGL-1 has at most two or three N-glycans that are not required for binding to P-selectin [11]. Treatment with sialidases indicated that α2,3-linked sialic acid is required for P-selectin binding, indicating that PSGL-1 expresses functional sialylated O-glycans. The glycoprotein was found to contain the sLex antigen and to have many sialylated, clustered O-glycans that render it susceptible to cleavage with O-sialoglycoprotein endopeptidase [12]. Treatment of intact myeloid cells with O-sialoglycoprotein endopeptidase eliminates the high-affinity binding sites for P-selectin without affecting overall surface expression of sLex [12, 13]. Antibody blocking studies and genetic deletion of PSGL-1 demonstrate that PSGL-1 is the dominant ligand for P- and L-selectins on leukocytes. Studies with synthetic glycosulfopeptides which mimic the N-terminal domain of PSGL-1, indicated that P-selectin binds in a stereo-specific manner to the amino terminal of PSGL-1 through recognition of a tripartite domain containing tyrosine sulfate residues, adjacent peptide determinants, and fucose, galactose and sialic acid residues on a core-2 O-glycan [14, 15]. The crystal structure of P-selectin complexed with a glycosulfopeptide derived from PSGL-1 revealed a broad shallow binding interface [8]. The Ca2+-dependent interactions with sLex were augmented by Ca2+-independent contacts with tyrosine sulfate and other amino acids. This explains why P-selectin binds with higher affinity to PSGL-1 than to sLex alone.
The Primary Structure of PSGL-1.
A cDNA encoding PSGL-1 was isolated from a human HL-60 cell library by expression cloning using COS cells that were panned on immobilized P-selectin [16]. Functional expression of PSGL-1 in COS cells required cotransfection with an α1,3 fucosyltransferase, and confirmed earlier observations that both α1,3 fucosylation and α2,3 sialylation of surface glycoproteins are required for binding to P-selectin [17]. The deduced amino acid sequence of PSGL-1 (SEQ ID NO:1) reveals a type 1 membrane protein of 402 amino acids. It has an NH2-terminal signal peptide, a propeptide that is cleaved by paired basic amino acid converting enzymes. The extracellular domain of the mature protein begins at residue 42 and has the hallmarks of a mucin. It is rich in serines, threonines, and prolines, and includes 15 decameric repeats. Three NH2-terminal tyrosines at residues 46, 48, and 51 are located in an anionic consensus sequence that favors tyrosine sulfation. There is a single extracellular cysteine located at the junction of the transmembrane domain, which is followed by a cytoplasmic domain of 69 residues. The cDNA for murine PSGL-1 reveals a protein of similar size to the human protein. Murine PSGL-1 also has a signal peptide, a propeptide, and a single cysteine near the transmembrane domain [18]. Furthermore, murine PSGL-1 has an anionic NH2-terminal sequence with two rather than three tyrosines. The sequences of the murine and human transmembrane and cytoplasmic domains are highly conserved, implying important functions. The murine extracellular domain, although rich in serines, threonines, and prolines, has only 10 decameric repeats, and shares little sequence similarity with the human protein. A single exon encodes the open reading frame in both the human and murine PSGL-1 genes [18, 19]. The sequence of PSGL-1 in most human leukocytes has an additional decameric repeat not found in the protein from HL-60 cells and other cell lines [19, 20]. Human PSGL-1 is sulfated [21-23], but the sulfate is present exclusively on tyrosine residues rather than on O-glycans [24, 21]. Sulfation occurs on one or more of the three clustered tyrosines at residues 46, 48, and 51 of SEQ ID NO:1 [22, 23]. Enzymatic removal of sulfate [21], blockade of sulfate synthesis [22, 23], proteolytic removal of an NH2-terminal fragment containing the three clustered tyrosines [25], or replacement of the tyrosines with phenylalanines [22, 23, 26] eliminates binding of PSGL-1 to P-selectin. Other structural features of PSGL-1 may also be important for optimal binding to P-selectin. The acidic residues surrounding the tyrosines may favor binding, although they are not sufficient in the absence of tyrosine sulfate.
PSGL-1 Binding to L- and E-Selectins.
PSGL-1 has also been shown to bind to both L-selectin [27-29] and E-selectin [16, 30-32]. Binding of PSGL-1 to L-selectin is blocked by the mAb PL1 [27-29, 33], by enzymatic removal of the NH2-terminal clustered tyrosines [29], or by the prevention of sulfate synthesis [29]. These results suggest that L- and P-selectins bind to a similar NH2-terminal region of PSGL-1 that requires both tyrosine sulfate and O-glycan(s). L-selectin binds to a group of mucins expressed on lymph node high endothelial venules (HEVs) and on some activated endothelial cells. These mucins include CD34, GlycCAM-1 and podocalyxin. PSGL-1 binds much differently to E-selectin than to P- or L-selectin. Core-2, sialylated and fucosylated O-glycans are required for binding to E-selectin [34], but tyrosine sulfation is not required [22, 23, 34]. E-selectin also binds to the NH2-terminal region of PSGL-1 [30, 35], although with lower affinity than does P-selectin [30]. The anti-PSGL-1 mAb PL1 has little or no effect on binding of PSGL-1 to E-selectin. This and other data indicate that E-selectin also binds to one or more still uncharacterized sites on PSGL-1 [35, 36]. Genetic deletion of PSGL-1 in mice impairs leukocyte tethering to E-selectin in vitro and in vivo [37]. Combined with the data on P- and L-selectin, this establishes that PSGL-1 is a physiologically relevant glycoprotein ligand for all three selectins.
The Tissue Distribution of PSGL-1.
Northern blot analysis indicated that mRNA for PSGL-1 is expressed in many human and murine organs, but did not indicate the specific cells in which it is expressed [16, 18]. Flow cytometric and immunocytochemical analysis of multiple human tissues with the anti-PSGL-1 mAbs PL1 or PL2 revealed that the PSGL-1 core protein is expressed primarily in hematopoietic cells [20, 38]. In bone marrow it is expressed on myeloid cells at many stages of maturation, but not on erythroid cells, megakaryocytes, or platelets. PSGL-1 is expressed on virtually all leukocytes, but at lower levels on B cells. P-selectin binds to PSGL-1 on all myeloid cells [20]. However, it binds to PSGL-1 on only a subset of T cells [20, 39]; most of these are memory cells [40] and they may be predominantly γ/δ cells [41]. PSGL-1 is expressed on circulating dendritic cells, on tissue monocyte derived dendritic cells, and on some dendritic cells in lymphoid organs; the function of the protein in these cells is unknown. PSGL-1 is also expressed on some CD34+ stem cells [38], where it may bind P-selectin [42]. The PSGL-1 protein is also expressed on epithelial cells lining the fallopian tube and in some endothelial cells at sites of chronic inflammation [38] and in epithelial cells and lamina propria of intestinal mucosal lining [126]. It has been reported that P-selectin is present on the surface of porcine sperm, where it binds to a P-selectin ligand which may be related to PSGL-1, on the zona pellucida of porcine oocytes [43].
PSGL-1-Selectin Interactions Mediate Tethering and Rolling of Leukocytes Under Hydrodynamic Flow.
The functional significance of PSGL-1 interactions with the selectins has been identified. Under hydrodynamic flow, selectin-ligand interactions must form rapidly to facilitate tethering, and then dissociate rapidly to facilitate rolling. Selectin-ligand bonds must have mechanical strength so that shear forces do not significantly accelerate the rate of dissociation [1]. There are relatively few copies of PSGL-1 on leukocytes [13. 20], and PSGL-1 displays only a small fraction of the total sLex-containing glycans on the cell surface [12]. However, PSGL-1 is the only high affinity ligand for P-selectin on intact leukocytes [20], and PSGL-1 is the essential ligand for mediating adhesion of leukocytes to P-selectin. The anti-PSGL-1 mAb PL1 blocks tethering and rolling of human neutrophils, eosinophils, and mononuclear cells on P-selectin under flow [20, 44]. PL1 also abrogates adhesion of neutrophils and monocytes to P-selectin under static conditions [20, 45, 46]. The PL1 mAb to an NH2-terminal epitope of murine PSGL-1 has been shown to block tethering and rolling of murine myeloid cells on P-selectin under flow [47]. Thus, interactions of PSGL-1 with P-selectin mediate adhesion of leukocytes to both activated endothelial cells and platelets under shear stress. The requirement for PSGL-1 to mediate leukocyte adhesion to P-selectin probably reflects, in part, its superior binding affinity relative to other ligands. The orientations of both PSGL-1 and P-selectin on the cell surface may also optimize their interactions under hydrodynamic flow. Both P-selectin and PSGL-1 are highly extended proteins, which project their NH2-terminal binding domains above most of the cell surface glycocalyx [13, 26]. Most of the O-glycans on PSGL-1 may function primarily to extend the NH2-terminal region above the plasma membrane. When expressed on transfected CHO cells, shortened P-selectin constructs with fewer short consensus repeats are much less effective than wild-type P-selectin in mediating tethering and rolling of neutrophils under flow [36]. PSGL-1 is also concentrated on microvillous tips [20, 48]. Thus, both the lengths and surface distributions of PSGL-1 and P-selectin may enhance rapid and specific interactions, yet minimize nonspecific repulsion between apposing cell surfaces. Upon neutrophil activation, PSGL-1 undergoes a cytoskeletal-dependent redistribution to the uropods of polarized cells [48-50]. This redistribution is associated with weakening of adhesion to P-selectin and transfer of adhesive control to integrins [49, 50]. Like PSGL-1, L-selectin is also concentrated on the tips of microvilli [51]. Leukocytes use L-selectin to roll on adherent leukocytes [52] or to initiate leukocyte aggregation [53]. Leukocyte-leukocyte interactions lead to secondary tethering of leukocytes to a P- or E-selectin surface, a potential mechanism for amplifying leukocyte recruitment to the vessel wall under shear forces [27, 54]. Flowing leukocytes roll on purified PSGL-1; this interaction is blocked by PL1 and by mAbs to L-selectin [27]. Furthermore, PL1 significantly inhibits the L-selectin-dependent rolling of neutrophils on adherent neutrophils [27] and the L-selectin-dependent aggregation of stirred neutrophils [33]. These data suggest that PSGL-1 is an important ligand for L-selectin under at least some conditions. However, there are L-selectin ligands other than PSGL-1 that participate in leukocyte-leukocyte contacts [27, 54, 55]. PSGL-1 may be one of only a few glycoproteins in human leukocyte lysates that binds well to E-selectin [31, 56]. Microspheres coated with recombinant PSGL-1 also roll on immobilized E-selectin under shear forces [35]. However, it is not clear whether PSGL-1 has any significant function for adhesion of leukocytes to E-selectin and this remains to be demonstrated. PL1 partially reduces accumulation of rolling neutrophils on E-selectin under flow [36]. But this effect occurs indirectly through inhibition of L-selectin-PSGL-1 interactions between neutrophils, thus reducing secondary tethering of neutrophils to E-selectin [36]. PL1 blocks primary tethering of flowing leukocytes to P-selectin but not to E-selectin [36, 54]. Human K562 cells transfected with FTVII roll on E-selectin in the absence of PSGL-1 [57]. Conversely, eosinophils, which express PSGL-1 but express relatively little total sLex, tether and roll much less efficiently on E-selectin than on P-selectin [44, 58]. Together, these data suggest that E-selectin must bind to ligands other than PSGL-1 to mediate leukocyte attachment under flow.
Signaling Through PSGL-1.
In the multistep model of leukocyte recruitment, leukocytes rolling on endothelial cells or platelets encounter regionally presented chemokines and lipid autacoids that stimulate the leukocytes to develop integrin-dependent firm adhesion and other responses. However, signals may also be directly transmitted through adhesion molecules [3]. The available data suggest that binding of P-selectin to PSGL-1 on leukocytes generates signals that must be integrated with those from other activators to elicit most effector responses [3]. In the best studied examples, monocytes mobilize the transcription factor NFκB and synthesize the cytokines TNFα and monocyte chemotactic protein-1 (MCP-1) when the cells adhere to immobilized P-selectin and platelet-activating factor, but not to either molecule alone [45]. Monocytes secrete a different profile of cytokines when they are exposed to P-selectin and the platelet derived chemokine, RANTES, but not to either protein alone [46]. Under some conditions, cooperative signaling through PSGL-1 and receptors for conventional activators may also generate other leukocyte responses [3, 59]. Adhesion of T cells to P-selectin was reported to induce tyrosine phosphorylation of the pp125 focal adhesion kinase (FAK), although the role of PSGL-1 in this event was not directly tested [60]. pp125 FAK has not been detected in human myeloid cells [61]. However, engagement of PSGL-1 with bivalent mAbs or immobilized P-selectin induces rapid tyrosine phosphorylation of other proteins in human neutrophils [62]. These include the ERK family of mitogen-activated protein kinases, which are activated by PSGL-1 engagement. Engagement of PSGL-1 with mAbs is sufficient to stimulate neutrophils to secrete IL-8. This secretion is blocked by a tyrosine kinase inhibitor, suggesting that tyrosine phosphorylation propagated through PSGL-1 may be physiologically important [62]. Cross-linking of L-selectin also rapidly transmits signals into both myeloid and lymphoid cells [63-68]. Thus, binding of L-selectin to PSGL-1 during leukocyte-leukocyte interactions may transmit bidirectional, potentially cooperative, signals during the earliest phases of leukocyte recruitment.
Physiological and Pathological Functions of PSGL-1-Selectin Interactions.
Recent in vivo studies have confirmed the predictions from in vitro experiments that PSGL-1 is a physiologically important selectin ligand. Anti-PSGL-1 mAbs inhibit rolling of both human and murine leukocytes on P-selectin expressed in postcapillary venules in vivo [47-69]. Polyclonal antibodies directed to the NH2-terminal segment of murine PSGL-1 specifically inhibit the recruitment of T helper 1 lymphocytes in a delayed-type hypersensitivity model [47]. A mAb to the NH2-terminal region of murine PSGL-1 also inhibits accumulation of neutrophils into chemically inflamed peritoneum [47]. The degree of inhibition is comparable to that observed with a mAb to P-selectin. More complete inhibition is observed with the combined use of mAbs to both PSGL-1 and P-selectin, suggesting that PSGL-1 interacts with at least one other molecule. An obvious candidate is L-selectin, given the in vitro evidence that binding of L-selectin to PSGL-1 mediates leukocyte-leukocyte interactions. Since PSGL-1 promotes adhesive interactions through both P- and L-selectins, it is almost certain to contribute to pathological leukocyte recruitment in a variety of inflammatory and thrombotic disorders in which P- and L-selectins have been implicated previously [6]. This suggests that mAbs to PSGL-1, soluble forms of PSGL-1, and other inhibitors of PSGL-1 function are potentially useful anti-inflammatory drugs in such conditions. In support of this concept, infusion of recombinant soluble PSGL-1 potently inhibits leukocyte infiltration and parenchymal damage in rat kidneys subjected to ischemia and reperfusion. Proteolytic removal of the NH2-terminal region of PSGL-1 abrogates its protective effects [70]. This implies that soluble PSGL-1 blocks adhesion of leukocytes to P-selectin and perhaps to L-selectin in this model. In other pathological states, soluble PSGL-1 may also be an effective E-selectin inhibitor even if PSGL-1 on leukocyte surfaces is not a dominant ligand for E-selectin. Use of a mAb blocking P- and E-selectin may not be supported by data in double P/E−/− knock out mice that showed increased susceptibility to infection an altered hematopoiesis [71].
Development of Anti-PSGL-1 Antibodies and Characterization of their Binding Epitopes on PSGL-1.
Several function-blocking mouse monoclonal antibodies to human PSGL-1 have been developed. A mouse monoclonal antibody named PL1 was developed using standard hybridoma technology by immunization of mice with PSGL-1 from human neutrophils [34]. PL1 was shown to bind a 14 amino acid epitope encompassing residues 49-62 of the native human protein (SEQ ID NO:1) using linear epitope mapping with overlapping octamer peptides spanning residues 19-77 of PSGL-1. PL1 was shown to block leukocyte adhesion to P-selectin in static adhesion assays and under flow [20]. Another anti-human-PSGL-1 antibody named KPL-1 was developed by immunization of mice with a recombinant form of PSGL-1. KPL1 inhibited interactions between P-selectin and purified CD4 T cells and neutrophils in flow assays, between lymphoid cells transfected with L-selectin and COS cells expressing PSGL-1, but did not block interactions of P-selectin or neutrophils on E-selectin [72]. KPL1 was subsequently shown to bind to a 17mer synthetic peptide encompassed by the binding domain of PL1 [73]. Another antibody, termed RR2r3s4-1, was engineered as a fully human antibody from a single chain Fv which had been isolated from a pool of PSGL-1 binders identified from a yeast surface display non-immune library [74, 125]. RR2r3s4-1 blocked neutrophil adhesion under flow and was shown to be specific for human, but not murine, PSGL-1.
Recent studies have also shown that PSGL-1 plays a dual function role in that along with its binding of selectins, PSGL-1 also interacts with chemokines to facilitate homing of T cells to secondary lymphoid organs [75-76].
Chemokines.
Chemokines are highly basic proteins consisting of 70-125 amino acids with molecular masses ranging from 6-14 kD [77, 78]. To date over 50 chemokines have been identified. The superfamily of chemokines is subclassified on the basis of the arrangement of cysteine residues located in the N-terminal region, as designated C, CC, CXC, and CX3C members, in which C represents the number of cysteine residues in the N-terminal region and X denotes the number of intervening amino acids in between the first two cysteines [77, 79, 80]. The CXC subfamily is sometimes further classified into ELR+ and ELR types based on the presence or absence of a triplet amino acid motif (Glu-Leu-Arg) that precedes the first cysteine residue in the primary amino acid sequences of these chemokines. The presence of this motif imparts an angiogenic function to this subset of CXC chemokines, while the ELR-chemokines have angiostatic properties [81], with the exception of SDF-1 which is angiogenic [82]. In general, the chemokines attract distinct classes of leukocytes: CC chemokines attract one or more classes of mononuclear cells, eosinophils and basophils; ELR+CXC chemokines attract neutrophils; ELR−CXC chemokines attract lymphocytes; C chemokine (lymphotactin) attracts T cells and CX3C chemokine (fractalkine) acts on T cells, natural killer cells and monocytes [83]. Chemokines are produced by a variety of cell types either constitutively or in response to inflammatory stimuli. Chemokines can be broadly divided into homeostatic and inflammatory categories based on their expression pattern and function in the immune system [78, 80]. The homeostatic chemokines are generally those that are “constitutively” expressed. They are involved in homeostatic lymphocyte and dendritic cell (DC) trafficking and lymphoid tissue organogenesis. The “inflammatory” chemokines are upregulated by proinflammatory stimuli and help orchestrate innate and adaptive immune responses. Although most chemokines are present in soluble forms and some may be associated with glucosaminoglycan moieties on the cell surface, two of the chemokines namely CX3CL1 (fractalkine) and CXCL16, have a natural mucin stalk that adheres onto the membrane of the cells that produce them [84, 85]. Their “chemokine” domain is located at the N-terminus of the mucin stalk and can be released by metalloproteinase cleavage. While the soluble, released chemokine domain of CX3CL1 and CXCL16 functions similarly to other secreted chemokines, their membrane bound forms play an important role in mediating leukocyte-endothelial cell adhesion and extravasation. Chemokines exert their biological effects by binding to G protein-coupled cell surface receptors. Nineteen chemokine receptors have been cloned so far [80, 86], including six CXC receptors (CXCR1-6), eleven CC receptors (CCR1-11), one CX3C (CX3CR1) and one C receptor (XCR1). Chemokine and receptor interactions vary widely in terms of selectivity. Some chemokines bind only one receptor and vice versa, such as the interactions of CXCR4 with CXCL12 (SDF-1), CXCR5 with CXCL13 (BCA-1), CXCR6 with CXCL16, CCR6 with CCL20 (LARC), and CCR9 with CCL25 (TECK). However, there is also redundancy in chemokine and receptor interactions since some chemokines bind more than one receptor and many receptors recognize more than one chemokine. For example, chemokine CCL5 (RANTES) has been shown to bind at least CCR1, CCR3 and CCR5, while CCR3 also binds CXCL1 1 (eotaxin), CCL24 (eotaxin-2), CCL26 (eotaxin-3), CCL8 (MCP-2), CCL7 (MCP-3), and CCL13 (MCP-4). Furthermore, two of the chemokine receptor-like proteins, the Duffy antigen receptor for chemokines (DARC) and D6, promiscuously bind many of the CXC and CC chemokines with equal affinity [87-89], but without being activated, presumably acting as sinks that sequester inflammatory chemokines.
Leukocyte Trafficking and Homing.
Chemokines control lymphocyte recirculation in immune system homeostasis, as well as in the activation dependent and tissue-selective trafficking of effector and memory lymphocytes. Lymphocyte homing to lymphoid and nonlymphoid tissues and recirculation between secondary lymphoid organs critically depend on the chemokines present in different sites. CCL19 and CCL21 (which bind to CCR7), and CXCL13 (which binds to CXCR5), are expressed in the lymphatic vessels, high endothelial venules (HEVs) and secondary lymphoid organs, and promote the entry of antigen-presenting cells (APCs), T cells and B cells into these organs [90]. Resident DC precursors in peripheral tissues phagocytose microorganisms or cell debris and are activated by pathogens or antigens. These cells then start to mature and express CCR7 which enables them to migrate in response to CCR7 ligands into the draining lymph nodes via the lymphatic vessels, and to infiltrate the T-cell zones where they present processed antigen epitopes to T cells. In contrast to DCs, B cells and naïve T cells enter lymph nodes through HEVs. The CCR7 ligands CCL19 and CCL21 produced by the endothelial cells of HEVs are transcytosed to the luminal surface and induce lymphocyte extravasation to the T-cell zones of the lymph nodes [91]. CCL19 produced by mature, inter-digitating DCs facilitates the “scanning” of DCs by naïve T cells in the lymphoid organs in search of their cognate antigens. B cells express CXCR5 and the ligand CXCL13 is produced by follicular stromal cells in lymph nodes. B cells activated by T cells proliferate in the follicles, giving rise to germinal centers (GC). Activated T cells expressing CXCR5 may also enter the follicles to participate in the T-B interaction. In addition, CCL19 and CCL21 are responsible for the proper positioning of lymphocytes within distinct microenvironments of lymphoid organs. For instance, CCL19 and CCL21, expressed by DCs and stromal cells retain T cells within the T-cell zones of secondary lymphoid organs. On the other hand, CXCL13 expressed by follicular DCs and stromal cells in follicles attracts B cells and some of the T cell subsets into the B-cell areas. Furthermore, the capacity of B cells to respond to CCR7 as well as CXCR5 ligands controls the position of B cells at the boundary of the follicles and T-cell zones in the spleen, where naïve, mature B cells interact with T cells that are newly activated in the adjacent zones [92, 93]. Non-activated B cells and T cells then leave the secondary lymphoid organs via the efferent lymphatics.
Inflammation.
A central feature of inflammatory diseases is the migration of leukocytes from the circulation, across the endothelium and the basement membrane, and into the affected tissue. The mechanism of extravasation is induced by chemokines (chemoattractant cytokines), which as noted above are a family of proinflammatory mediators produced at the inflammatory site. As part of the migration process, circulating leukocytes must first adhere to the luminal surface of the endothelium. According to the current paradigm, this interaction involves the sequential engagement of leukocyte and endothelial adhesion molecules. First, selectins and their glycoprotein and carbohydrate counterligands mediate leukocyte tethering and rolling. Then, leukocyte integrins and their ligands, including immunoglobulin-like intercellular adhesion molecules, mediate firm leukocyte adhesion. Chemokines play a role in firm adhesion by activating integrins on the leukocyte cell surface. The leukocytes are directed by chemoattractant gradients to migrate across the endothelium, and through the extracellular matrix into the tissue.
The events that lead to an inflammatory response are characterized by recognition of the site of injury by inflammatory cells, recruitment of specific leukocyte subpopulations, removal of offending microbial invaders, “debridement” of injured cells/tissues, and wound repair. Chemokines have been shown to participate in and control the process of a number of acute and chronic inflammatory conditions by promoting the infiltration and activation of inflammatory cells into injured or infected tissues [94].
Several of the CC chemokines including CCL3 (MIP-1α) and CCL5 (RANTES) are expressed in sepsis and exert proinflammatory effects by mediating organ specific leukocyte influx and activation [95, 96]. Members of the CXC chemokines are implicated in the pathogenesis of systemic inflammatory response [97, 98]. In bacterial pneumonia, CXC chemokine-mediated elicitation of neutrophils is beneficial and necessary for clearance of invading microorganisms [98]. To support this notion, over expression of KC, a murine homologue of human CXCL1 (GRO-α), specifically in the lung, enhances resistance to Klebsiella pneumonia [99]. In asthma, the submucosa of small airways is infiltrated by mononuclear, eosinophil and mast cells causing mucous gland hyperplasia and subepithelial fibrosis. Animal models of allergic airway inflammation and asthmatic patients imply a key role for chemokines in regulating lung inflammation [100]. The kinetics of production of CCL2, CCL11, CCL17 and CCL22 correlates with the recruitment in airways of specific leukocyte subsets expressing the receptors for these chemokines [101]. Chronic obstructive pulmonary disease (COPD) is characterized by progressive development of airflow limitation caused by chronic inflammation with increased recruitment of neutrophils, macrophages and IFN-γ-producing CD8+ T cells in the lung. In COPD patients, the levels of CXCL8 and CXCL10 are increased and correlate with the degree of infiltration by neutrophils and CD8+ T cells that produce IFN-γ. The lung-infiltrating T cells express CXCR3, the receptor for CXCL10 [102], suggesting that CXCR3 may mediate the recruitment of pathogenic Th1 cells into chronically inflamed lungs. Neutralization of CXCL10 also appears to inhibit allergic airway inflammation [103]. Thus, in addition to many other chemokines, CXCR3 and its ligands participate in lung inflammation that is not necessarily dominated by Th1 response. Atherosclerosis is widely accepted as an inflammatory disease [104], in which chemokines play a central role in leukocyte recruitment, angiogenesis, and more intriguingly in the proliferation of vascular smooth muscle cells and their migration into plaques [105]. Atherosclerotic lesions express a number of chemokines including CCL2, CCL3, CCL4, CCL5, CCL11 and CXCL8. The cellular sources of chemokines within atherosclerotic lesion are multiple and include endothelial cells, smooth muscle cells and infiltrating leukocytes. There is overwhelming evidence to support the involvement of CCL2/CCR2 chemokine-receptor pair in atherosclerosis. CCL2 is essential for monocyte recruitment, has angiogenic activity and also causes smooth muscle cell proliferation and migration. Many factors known to promote atherosclerosis such as plasma cholesterol, hypertension and diabetes, stimulate chemokine release by atheromatous lesions. Adhesion of leukocytes to endothelial cells also augments chemokine release in the pathogenic process of atherosclerosis. Therefore, chemokines and receptors become important molecular targets for circumventing the formation and development of atherosclerotic lesions. In human, CX3CR1 gene polymorphism in the coding region confers individuals with protection against atherosclerosis [106, 107]. An M280 mutation in CX3CR1 results in loss of function of CX3CR1 since cells transfected with this mutant receptor exhibit a markedly reduced response to CX3CR1 ligand CX3CL1 [108]. When ApoE transgenic mice, an atherosclerosis model, were crossed with CX3CR1−/− mice, the severity of atherosclerotic lesion was significantly reduced with lower macrophage infiltration. This provides an excellent example of the importance of a functional chemokine receptor in contributing to the progression of atherosclerosis. Rheumatoid arthritis (RA) is characterized by a mixed Th1-type inflammatory cell infiltration (Th1 cells, neutrophils, monocytes) in synovial space of the joints [109], in association with cartilage destruction and bone remodeling. Chemokines produced in the inflamed joints attract leukocytes across the endothelial barrier to initiate and maintain active RA [110, 111]. Among CXC chemokines, high concentrations of CXCL8, CXCL5, CXCL1 are detected in the sera, synovial fluids, and synovial tissues of RA patients [109, 110]. These chemokines attract neutrophils and promote angiogenesis [109, 110]. Abundant production of CC chemokines CCL2, CCL3 and CCL5 which attract mainly monocytes is also found in RA [109, 110]. On the other hand, CXCL12 expressed in the rheumatoid synovium, recruits CD4 memory T cells, which express increased levels of CXCR4, at the RA site [111]. CXCL12 also blocks T cells from undergoing activation-induced apoptosis, thus further increasing the accumulation of T cells in the rheumatoid synovium. Interestingly, CXCL12 may induce the migration of DCs from blood stream into the rheumatoid area, implying its potential role in amplifying a detrimental autoimmune response. Multiple sclerosis (MS) as a chronic inflammatory demyelinating disorder of the central nervous system (CNS) is thought to be caused by an autoimmune response directed against self-myelin-associated antigens. The immune cells infiltrate in CNS lesions of MS patients consist of CD4, CD8 T cells and macrophages [112]. Many chemokines are detected in active lesions in the CNS of MS patients and the cerebrospinal fluids of relapsing patients contain elevated levels of CCL3 [113, 114]. In MS, infiltrating macrophages express CCR2 and CCR5, while T cells and reactive astrocytes in active lesions express CXCR3 and CCR5 [115, 116]. Similar chemokine expression patterns are found in experimental autoimmune encephalomyelitis (EAE), an animal model more related to MS. In EAE, increased expression of CCL2, CCL3, CCL4, CCL5 and CXCL10 correlates with the severity of the disease ([117]). Neutralizing antibodies to selected chemokines either inhibit the onset or reduce the severity of the EAE [118, 119]. A more definitive correlation between chemokines and EAE was established by experiments with CCR1- and CCR2-deficient mice, in which a reduction in disease incidence and severity were clearly documented [120, 121]. A link between chemokines and Crohn's Disease has also been established. The expression of chemokines CCL-19 and CCL-21 have been shown to be upregulated in the colon tissue, secondary lymphoid tissue and mesenteric lymph nodes derived from patients with Crohns disease [122, 123]. Further, the CCR7 receptor is also upregulated on dendritic cells in the colonic tissue of these patients which interact with T cells resulting in activation and proliferation. This increased expression of chemokines and chemokine receptors leads to increased retention of dendritic cells in colon tissue resulting in the formation of tertiary lymphoid tissue formation in the bowel wall which maintains the autoimmune inflammation in Crohn's disease [122, 123].
Clearly, the development of human- (and primate-) compatible monoclonal antibodies which block the chemokine mediated migration of leukocytes into, and their P-selectin mediated adhesion and rolling to cells in areas of inflammation and which have reduced immunogenicity would be of great value.